Numerous studies, including those in the area of breast cancer research, have begun to focus on targeting the radiation/chemo resistance of tumor initiating cells (TICs) that are thought to be responsible for tumor initiation, progression, and metastasis. However, the role of the putative "niche", a microenvironment that helps TICs maintain their functional activity to self-renew and to differentiate in breast cancer is largely unknown. Using in vivo limiting dilution transplantation experiments, and microarray analysis, a subpopulation of TICs as well as a subpopulation of cells in these tumors with mesenchymal features have been identified. These latter 'mesenchymal-like" cells display properties of cells, which may comprise a putative tumor stem cell "niche" by promoting TIC self-renewal in vitro. Within this mesenchyma-like subpopulation, increased expression genes encoding cytokines, chemokines, growth factors, and secretory Wnt proteins was observed. Importantly, these same factors also have been reported to function as niche components in various tissues. Accordingly, a p53 null syngeneic transplantation mouse model, which maintains an intact immune system and an appropriate microenvironment, will be employed to investigate the interaction between the niche cells and the TICs. By applying both in vitro genetic approaches (such as short hairpin RNA (shRNA) knockdown and lentivirus transduction) as well as in vivo functional assays (limiting dilution transplantation), we should be able to elucidate the critical molecular interactions between niche and TICs. Three aims are proposed, 1) to determine the functional interaction between niche cells with TICs through in vitro and in vivo assays;2) to elucidate the mechanisms regulating the supportive role of niche cells on TICs;and 3) to determine the role of niche cells in human xenograft breast cancer models. These studies should, therefore, help us better understand the role niche cells may play in the deregulation of self renewal and differentiation of the TICs. Insights into niche-TIC interactions may provide new therapeutic targets for drug development with the goal of both preventing breast cancer and reducing metastasis. I will be using new experimental approaches to enhance my research skills. This is essential for the achievement of my long term goal of being an independent investigator in an academic institution to investigate the interaction between stem cells and their microenvironment. Dr. Rosen is an internationally recognized leader in the areas of mammary gland biology and breast cancer. He has made significant contributions to our current understanding of breast cancer in the area of molecular biology of mammary gland gene expression, and mammary gland stem cells. Baylor College of Medicine, Department of Molecular and Cellular Biology and Breast Center maintain a variety of core facilities, compressive training programs, and a highly collaborative atmosphere with over 30 mammary gland biologists/breast cancer researchers. Thus, I believe this will provide me an outstanding training environment that will facilitate my transition to become an independent scientist. PUBLIC HEALTH RELEVANCE: The functional interaction between the cancer stem cells (tumor-initiating cells, TICs), and their microenvironment, the "niche" will be investigated using a p53 null syngeneic transplantation mouse model, as well as an established human breast cancer patient-derived xenograft model. Both in vivo and in vitro assays will be applied in studying how the components of the niche microenvironment affect stem cells, and how the deregulation of these interactions via genetic or epigenetic alterations may lead to tumor initiation. Insights into niche-TIC interactions may provide new therapeutic targets for drug development with the goal of both preventing breast cancer and reducing metastasis.